The Genetic Screening And RTN3 Functional Research In Cardiovascular Anomalies

Background: Cardiovascular system consists of heart and blood vessels including arteries,veins,and capillaries.A series of diseases in cardiovascular system triggered by external environment,somebody’s own condition,and/or genetic etiologies,are collectively called“cardiovascular diseases(CVD)”.Cardiac anomalies(such as congenital heart diseases [CHD],arrhythmia,cardiomyopathies)and vascular anomalies(including hypertension,pulmonary arterial hypertension [PAH],thoracic aortic dissection [TAD])are belong to CVD,which seriously affect human health and are the leading cause of death in the world.China is one of countries with the most CVD patients,where 40% death and early death are associated with CVD.Now,the pathogenic mechanism of CVD is not fully understood,while genetic factor,one of CVD causes,should be studied further.RTN3 belongs to reticulon family,which is a transmembrane protein mainly located in the endoplasmic reticulum and highly evolutionarily conservative.RTN3 has been reported to expression in the brain,peripheral nervous system,lung,liver,eye,testis,kidney,and lipid in mouse.Current research of RTN3 has focused on its role in the brain,lipid,and tumors.Our previous study found that knock out RTN3 and RTN1 resulted in abnormal cardiac development and stillbirth in mouse embryos,and a RTN3 variant was identified in a hypertensive family.However,the correlation between RTN3 and cardiovascular anomalies has not been reported.Purpose: 1)Carry out the genetic screening in CVD cases to identify their causative mutations and confirm the genotype-phenotype correlation;2)Investigate the correlation between RTN3 and dysarteriotony,and clarify the molecular mechanism;3)Explore the function and molecular mechanism of RTN3 in the heart by mice model.Methods: We recruited 225 patients with CVD,involved CHD,arrhythmia,cardiomyopathies,TAD,PAH,and hypertension,and used karyotype analysis,chromosomal microarray analysis(CMA),wholeexome sequencing(WES),and Sanger sequencing to identify their genetic etiologies.ELISA,WB,immunofluorescence,Co-IP,and RNAseq were performed to investigate the function and mechanism of RTN3 in the vessel and heart.Results: 1)We identified 9 chromosomal abnormalities,6 copy number variations(CNVs),and 25 gene mutations(including 15 novel mutations)in these 225 CVD cases,and verified the pathogenicity of some mutations in vitro,for example that GATA5 missense mutation(NM＿080473.4: c.830C>T,p.P277L)inhibited the nuclear translocation,and that MYBPC3 synonymous mutation(NM＿000256.3: c.24A>C,p.P8P)broke downstream splicing site.In addition,we detected a RTN3 variant in a hypertension case,whereas the correlation between RTN3 and cardiovascular anomalies has not been reported.To investigate the correlation,we performed the following studies.2)We found parts of hypertension patients with the increase of plasmatic RTN3,and RTN3 KO mice with lower blood pressure.Through cell and molecular experiments,we detected that RTN3 interacted with ceramide synthases CERS1,2,and 6 to inhibit ceramide synthesis,and participate in sphingosine 1-phospate(S1P)synthesis,thereby affecting blood pressure.3)In mouse models,we found that RTN3 deficiency inhibited FOXO3 A phosphorylation,and promoted FOXO3 A to transport into the nucleus,resulting in smaller cardiac cardiomyocyte size and reduced cardiac ejection fraction in two-month-old mice.Conclusions: Our investigation shows that RTN3 can control blood pressure and cardiomyocyte size,and illustrate its possible mechanism,suggesting that RTN3 may be a potential target for the diagnosis and treatment of hypertension and cardiomyopathy.Our identification in CVD patients detects novel mutations,expands the genetic spectrum of CVD,contributes to genetic counseling and molecular diagnosis of CVD,and deepen our understanding of CVD-related genetic factors.We perform function verification of the GATA5 missense mutation(c.830C>T,p.P277L),MYBPC3 synonymous mutation(c.24A>C,p.P8P),and CSRP3 nonsense mutation(c.364C>T,p.R122X),helping us to understand their pathogenic mechanism.We find that RTN3 can control blood pressure and cardiomyocyte size and preliminary clarify its mechanism,maybe a potential target for the diagnosis and treatment of hypertension and cardiomyopathy.